Probiotic bread and method of its production

The present invention refers to a process for preparing probiotic bread having a high content of viable probiotic bacteria, as well as a bread containing specific probiotic bacteria.

THE BACKGROUND OF THE INVENTION

Probiotics, that is viable microorganisms having scientifically documented positive health effects in man or animals, are today present in above all different dairy products such as sour milk, yoghurt and milk, in different types of beverages and soups such as for instance juices, fruit beverages, rose hip soup and blueberry soup, and as a powder, tablets or capsules.

A common bread on the other hand does not contain any microorganisms but is in principal quite sterile when it comes out of the oven, wherein it has been subjected to high heat so that the crumb of the bread for a period of time has had a temperature just below 100 ^° C. The bread is then allowed to cool, often on special cooling tracks and can then be subjected to recontamination by microorganisms on the surface of the bread. Said microorganisms, normally mould and yeast fungi, can subsequently, if the conditions are proper, grow and destroy the possibility of consumption of the bread. If bread is kept at ambient temperature the shelf life of the bread is in general about 10 to 14 days at the most.

Baking with sour dough is a very old method, above all involving advantages as to the elasticity of the bread dough in kneading and the ability to keep the carbon dioxide produced by the yeast, especially in rye dough, the aroma development in the bread dough and during the baking (for instance the Maillard reactions) and the shelf life of the baked bread as to fresh keeping and inhibitory effect on mould and yeast growth. To be able to prepare sour dough of a high and absolutely constant quality it is necessary to use a so called starter culture containing one or more well defined microorganisms. It is also necessary to prevent growth of the natural flora of microorganisms normally contained in the flour of the sour dough. The common process is then to mix the sour dough as an ingredient into the final bread dough before baking the bread in the oven, which result in all the sour dough bacteria being killed.

The problem with preparing bread containing a uniform distribution of probiotic bacteria is thus that if the bacteria are mixed into the bread dough before baking, they will be killed during the very baking process. In addition it is important that the content of probiotic

bacteria in the bread is not substantially changed during the shelf life of the bread of about 10 days when stored at ambient temperature, which temperature can differ depending on the time of the year from normally about 15°C to about 30°C, or for a shorter period of time at temperatures up to 40-45°C. The last mentioned temperatures can for instance prevail during transport on warm summer days.

PRIOR ART

WO 94/00019 discloses a baked product containing viable microorganisms and a method of preparing thereof. The actual microorganisms, yeast or bacteria, are said not to be able to propagate but rather to loose in viability during the period between preparation and consumption. In order to prepare a baked product having a desiderate number of microorganisms it is thus necessary to add said microorganisms to a higher content than what is desirable in the final baked product. Four different species, the strains of which have not been specified, have been used in the examples carried out.

DESCRIPTION OF THE INVENTION

The present invention refers to a process for the preparation of bread having a high content of viable probiotic bacteria, and a bread having a content thereof.

The invention refers to a process for preparing bread containing viable probiotic bacteria, wherein a bread is baked and allowed to cool a liquid formulation of probiotic bacteria is added to the crumb of the bread via a liquid sour dough to a content of viable bacteria of at least 1 x 10 ⁶ CFU/g bread. The invention especially refers to a process for preparing bread containing viable probiotic bacteria, wherein the probiotic bacteria are added to the crumb of the bread in an amount of at least 1 x 10 ⁷ CFU/g bread, especially at least 5 x 10 ⁷ CFU/g bread. CFU refers to colony forming units.

When adding probiotic bacteria to a bread there is a great risk that the number of bacteria decreases strongly and that the probiotic effect of the final bread will thus not appear. Instead, in order to adapt the probiotic bacterium to the environment in a crumb of a bread, the bacterium is first added to a dough in which optimal conditions are created for bringing about a growth of the bacterium at the same time as the pH value is declining. The sour dough is then added to the final bread. Having adapted the probiotic bacterium in this way to an

environment wherein flour substantially is the nutriment for the bacterium, it was found that there will instead be a growth in the bread during the first time of the storage. This implies great economical advantages, as a considerably less amount of bacteria has to be added to the crumb compared to if the number of bacteria decreases, in which case a higher number of bacteria has to be added to the bread. In addition an active probiotic bacterium is considerably more effective than one that is not well and thus will need some time in the intestines of the person consuming the bread to repair the cells before functioning to 100 %.

An additional advantage of adding the bacteria via a sour dough is that the probiotic bacterium will then be adapted to an environment having a low pH value, below pH 4, which means that it will also have a better ability to survive during the passage from mouth to intestines, where the bacterium for instance passes the low pH of the stomach.

The liquid formulation in which the probiotic bacteria are added is a liquid sour dough. Such a sour dough mainly contains water, flour and probiotic bacteria to a number of at least 1x10 ⁷ CFU/g. The flour can for instance be flour from cereals, such as rye meal or rye flour, pure sifted rye flour, sifted bakery flour or wheat flour, whole wheat flour, barley flour, oat flour, corn flour etc. Probiotic sour dough is a sour dough which adds to a bread both a tastyness and a preserving effect due to its content of above all different acids and preferably lactic acid, and antibiotic substances such as for instance different bacteriocines. As the probiotic sour dough preferably is added to the bread after baking, the bread will, in addition to the tastyness and preserving effect, also be provided with viable probiotic bacteria, which in a decisive way distinguishes the effect of this sour dough from the effect of traditional sour dough. As an example of such a sour dough can be mentioned the viable sour dough product described in SE 515 569.

Bread refers in this context to all bakery products the main ingredients of which are cereals, for instance oats, barley, rye, wheat or corn, and which undergoes a refinement process normally comprising both a raising step with yeast or a chemical leavening agent and a heating step, baking in an oven. As examples of different products can be mentioned soft crumb bread, crisp bread (knackebrδd), tortillas, pizza bread, rusks, biscuits, muffins, white wheat bread and cakes. When the bread has been baked, removed from the oven and cooled the probiotic liquid formulation is added. This can be done in several different ways depending on the actual type of bread. One way for soft crumble breads is to add the liquid probiotic formulation, at the same time as the bread is sliced, directly on to the slices of bread for instance via the "saw-blades" slicing the bread. This can for instance be done via small holes

in the "saw-blade" or via a separate, similar means in a separate step directly after the slicing of the bread. Another way is to "inject" the probiotic formulation via needles or tubes of a small diameter. This can be done either via the long sides of the bread in which case the length of the needles should correspond to the breadth of the bread, or from one or both of the short sides of the bread. If it is done from the two short sides at the same time, the length of the needles/tubes should be somewhat longer than the length of half of the bread. It is then also of importance that the needles/tubes are located not to collide in the centre of the bread, but rather to be able to pass by each other for a short distance. If the "injection" takes place from just one short side of the bread the length of the needles/tubes should correspond to the length of the bread. In order to get a uniform distribution of the bacterial formulation in the bread it is of advantage if the formulation is added through more than one needle/tube. A suitable number of needles/tubes will be somewhere in between 10 and 30 on a surface of a section of the bread of 1 dm ² . Said techniques will give a homogenous distribution of the bacteria in the crumb of the bread and the slices of bread, respectively. The invention especially refers to a process, which is characterised in that the probiotic bacteria are added by injection either multifocally or continuously by means of needles.

After finished injection bread can preferably be stored for at least 12 hours, during which period the probiotic bacterium grows somewhat and is activated in the crumb of the bread. A bread prepared according to this process will then have a number of viable probiotic bacteria higher than 1x10 ⁷ CFU/g bread. After storing for 9 days at 25°C the number of viable bacteria is higher than 5x10 ⁶ CFU/g bread.

The invention especially refers to the use of bacteria having the ability to grow and produce acids in a dough system, in which the flour can be wheat, rye, barley, corn, sorghum as well as oats. For certain bacteria, for instance propionic acid bacteria, a specific carbon source has to be added to the dough for an effective growth to be obtained. In the case of for instance propionic acid bacteria about 2 % by weight of L-lactate (based on the content of flour) is added to the dough recipe. Depending on the bacterial strain, the properties of the flour and optional additives, the number of bacteria in the sour dough after the fermentation step will be in between 1 xl O ⁸ and 2x10 ⁹ CFU/g.

The selection of bacterium is of great importance for obtaining an effective probiotic effect of the bread. It is required that the bacterium has the ability to survive in a bread crumb and then preferably also to grow in a sour dough, and in addition it is of great importance that the probiotic bacterium has the ability to adhere to surfaces and preferably to

the mucous membrane of the intestines. One possibility to investigate this characteristic in the selected bacterium is to look at its ability of self-agglutination and adhesion to cells of baker's yeast. In a study by Adlerbert, I., Ahrne, S., Johansson, M.-L., Molin, G., Hansson, L.-A., and Wold, A. 1996, A mannose-specific adherence Mechanism in Lactobacillus plantarum conferring binding to the human colonic cell line HT-29; Applied and Environmental

Microbiology 62:2244-2251, it was shown that the ability to adhere to a cell of bakery yeast in a mannose-specific way correlates to the ability of the bacterium to adhere to human colonic cells (HT-29) via mannose-specific receptors. Such a characteristic is of course very interesting when it comes to the possibilities of the probiotic bacterium to compete with all the other intestinal bacteria present.

The probiotic bacteria to be added to the bread of the invention especially belong to one or more of the genera Lactobacillus, Leuconostoc, Pediococcus, Weisella, Bifidobacterium and Propionibacterium.

The probiotic bacteria specifically belong to one or more of the species Lactobacillus plantarum, L. casei, L. acidophilus, L. reuteri, L. rhamnosus (L. casei ssp. rhamnosus), L. delbrueckii, L. salivarius, L. brevis, L. johnsonii, L. spicheri and L. sanfranciscensis, Leuconostoc mesenteroides, Pediococcus pentosaceus, Bifidobacterium bifidum, Propionibacterium shermanii, P. jensenii, or P. freudenreichii.

According to a preferred aspect the process of the invention especially refers to the use of probiotic bacteria having the ability to grow in a liquid formulation based on cereals. Examples of strains having this characteristic are for instance the following:

Lactobacillus rhamnosus 271 has been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany on July 2, 1991 and received the accession number DSM 6594. Further information about this strain is given in inter alia WO 93/01823.

Lactobacillus plantarum 111 has been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany on August 21, 1998 and received the accession number DSM 12383. Further information about this strain is given in inter alia SE 515 569. In the process of the invention for preparing bread can especially be used the probiotic bacterium Lactobacillus rhamnosus 271 (DSM 6594).

Especially Lactobacillus plantarum Il 1 (DSM 12383) can also be used. According to a preferred aspect the process of the invention especially refers to the use of probiotic bacteria having the ability to agglutinate. As examples of strains having

this property and in addition the ability to grow in a liquid formulation based on cereals can be mentioned:

Lactobacillus plantarum 299, which has been deposited at the Deutsche

Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, on July 2, 1991 and received the accession number DSM 6595.

Lactobacillus plantarum 299v has also been deposited at the Deutsche

Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, and received the accession number DSM 9843.

The new bacteria Lactobacillus plantarum 17, 3R9, and SPl 58, as well as L. gasseri SOl 21 all have been isolated from different types of sour dough. Strain 17 is isolated from an Icelandic industrial sour dough, the strains 3R9 and SOl 21 from two different

Swedish industrial sour doughs, and strain SP 158 from a spontaneously started wheat sour dough at a laboratory. The strains have been isolated on the substrate MRS supplemented with 1 % yeast extract and 2 % maltose, except for SP 158 where the substrate was solely MRS. The strains have been characterised as to ability to ferment different carbon sources by means of API 50 trays and API CHL medium (API system, Montalieu Vercieu, France) and on RAPD (Randomly Amplified Polymorphic DNA), giving an "artificial fingerprint" of the genome of the bacterium. The isolates have been isolated three times before being tested on

API and RAPD and the results showed that the four strains differed from the type strains of the respective bacterial species. The bacterial strains have been deposited at the Deutsche

Sammlung von Mikroorganismen und Zellkulturen GmbH, on November 16, 2006 and been given the following accession numbers: Lactobacillus plantarum 17 (DSM 18787);

Lactobacillus plantarum 3R9 (DSM 18788); Lactobacillus plantarum SP158 (DSM 18789); and Lactobacillus gasseri SO121 (DSM 18790). Plasmid DNA was prepared according to Anderson, D., G., and McKay, L. L.

(Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Applied and Environmental Microbiology, 1983; 46: 549-552).

The plasmids were separated by electrophoresis on a 0.7 % agarose gel in Tris- borate buffer. DNA was then visualised by UV light after staining with ethidium bromide and the gel was photographed. The distance that DNA had moved on the gel was measured directly from the photo of the gel. As reference strain was used Escherichia coli V517

(Plasmid reference Center, Stanford, Kalifornien, USA).

The results showed inter alia differences between L. plantarum 299 (DSM

6595) and 299v (DSM 9843), having the same plasmid pattern, and the other strains of I.

plantarum as to plasmid patterns. Certain plasmid sizes were found in all strains, other plasmids, however, were unique for the respective strain.

The invention especially refers to a process for preparing bread containing viable probiotic bacteria, wherein the probiotic bacteria are one or more of the following strains:

Lactobacillus plantarum 299v (DSM 9843); Lactobacillus plantarum 299 (DSM 6595); Lactobacillus plantarum 17 (DSM 18787); Lactobacillus plantarum SP158 (DSM 18789); Lactobacillus plantarum 3R9 (DSM 18788); Lactobacillus gasseri SO121 (DSM 18790).

The probiotic bacteria should be added to the bread crumb in an amount giving a probiotically active amount of bacteria when ingesting the bread. An intake per day of 10 ⁹ CFU probiotic bacteria can be sufficient to provide a positive effect in the intestinal flora, but often it is desirable with an amount being one or a few powers often higher. A daily delivery of 1-5x10 ¹⁰ bacteria is normally used to give the desired effect. The delivered amount of bacteria of course also depends on the ability of the respective strain to survive the passage from mouth to intestines and its ability to remain in the intestinal system. If the strain is able to adhere to intestinal cells the positive effect will increase. 2-3 slices of bread per day correspond for instance to 80-120 g bread, which should give an intake of at least 10 ⁹ CFU/d. The amount of bacteria to be added to the bread will in this case be at least 8.3xlO ⁶ to 1.25xlO ⁷ CFU/g. For a higher intake it might be suitable with 5xlO ⁷ to 5xlO ⁸ CFU/g bread.

The invention also refers to a bread containing at least 1x10 ⁷ CFU viable probiotic bacteria having the ability to grow in a liquid formulation based on cereals and to agglutinate, per g.

The invention especially refers to a probiotic bread containing one or more probiotic bacteria selected from the group comprising Lactobacillus rhamnosus 271 (DSM 6594); Lactobacius plantarum 111 (DSM 12383); Lactobacillus plantarum 299v (DSM 9843); Lactobacillus plantarum 299 (DSM 6595); Lactobacillus plantarum 17 (DSM 18787); Lactobacillus plantarum SP158 (DSM 18789); Lactobacillus plantarum 3R9 (DSM 18788); and Lactobacillus gasseri SO121 (DSM 18790).

EXAMPLES

Example 1. Test of the activity of different bacteria in a water solution based on cereals

To find out if a bacterial strain has the ability to grow well and propagate, as well as to decrease the pH value in a dough, different bacterial strains were tested in a so called activity test. 225 g of water was mixed with 15O g of rye flour and the actual bacterial strain in an amount of 1x10 ⁷ CFU/g dough. The time that lapsed until pH had decreased to 4.0 was measured, as well as the pH value, the acid equivalent (S °, ml 0.1 N NaOH required to give a pH of 8.50 in a solution consisting of 10 g of dough and 90 g distilled water) and the number of viable bacteria in the mature sour dough CFU/g. The time of fermentation was 20 h and the fermentation temperature 30°C or what was optimal for the added starter culture. The result is presented in the table below.

Table 1

Bacterial Time to pH, 20 h S\ 20 h CFU/g, 20 h strain pH= 4 •O fh)

L acidophilus PT24 18.5 4.2 10.6 3 10 ⁸

L brevis A6 12.0 3.6 16.1 2 10 ⁹

L gasseri $O\2\ 10.5 3.5 21.5 5 10 ^s

L plantarum 17 10.0 3.6 14.5 9 10 ⁸

L plantarum Il 1 9.5 3.5 14.8 8 10 ⁸

L plantarum SPl 58 9.5 3.5 15.5 no ⁹

L plantarum 3R9 9.0 3.5 14.7 no ⁹

L plantarum 299v 10.5 3.5 15.1 2 10 ⁹

L rhamnosus 271 13.0 3.6 13.7 no ⁹

Lactococcus lactis > 20 5.2 4.8 < 1 10 ⁷

The results show that not all strains of so called lactic acid bacteria have the ability to grow well in a dough containing but water and flour.

Example 2. Test of the agglutinative ability of different bacteria

Certain bacterial strains have the ability to adhere to different types of surfaces. It could be a surface of glass or to the cell surface of the same bacterial strain as itself, self- agglutination, or to a baker's yeast cell of the species Saccharomyces cerevisiae. The property to self agglutinate means that the bacterium has a greater ability to be able to form bio films. This is of great interest when it comes to the survival of the bacterium and its growth in the final bread, which becomes much better. The biofilm is a kind of protective mechanism for the bacterium and bacteria easily form such a one around structures such as for instance fibres in the sour dough and the crumb of bread. This also implies that the bacteria are more protected during the passage from the mouth down through the stomach to the intestines and that a greater number with high activity reaches the final destination in the intestines.

As a test of the above characteristics and of the ability to bind mannose-specific to baker's yeast the method described by Adlerbert et al. (1996) was used. Bacteria having this characteristic have turned out to bind to mannose-specific receptors on the human intestinal mucosal membrane.

The assay involves mixing a drop containing baker's yeast dissolved in PBS solution (phosphate buffered saline solution) with another drop containing the bacterial culture dissolved into PBS solution on a microscope slide. The slide is then waddled back and forth and the number of turnings required to visualise a fine granular precipitate recorded. If no precipitate is visible after about 25-30 turnings the bacterial culture is deemed to be non yeast agglutinating. A similar test is then performed in which a drop of bacterial solution is mixed with a drop containing both baker's yeast and D-mannose and at the same time a drop of the bacterial solution is mixed with a drop only containing baker ^' s yeast. To verify a mannose-inhibited agglutination an optional agglutination should take place after more than at least 10 turnings when mannose is added compared to agglutination without mannose. Table 2

Bacterial SeIf- Binds to Binds mannc strain aεglutin. veast cell to veast cell

L. acidophilus no no no

L. brevis A6 no no no

L. gasseri SO121 yes no no

L. plantarum 17 yes no no

L. plantarum Il 1 no no no

L plantarum SPl 58 yes yes yes

L. plantarum 3R9 yes no no

L. plantarum 299 yes yes yes

L. plantarum 299v yes yes yes

L. rhamnosus 271 no no no

Lactococcus lactis no no no

The results show that only certain bacterial strains have the ability to adhere to surfaces. The invention especially refers to the use of bacteria having the ability of self-agglutination and/or binding to yeast cells and/or bindning to yeast cells in a mannose-specific way.

Example 3. Preparation of probiotic bread with Lactabacillus plantarum 299v

Sour dough is prepared using the probiotic starter culture L. plantarum 299v (DSM 9843), wherein a mixture of water, sifted bakery flour and an enzyme mixture (San Super 240 L (Novo Nordisk A/S, Bagsvaerd, Denmark)) is blended and heated in two steps to about 90°C. After a holding time of 60 minutes the mixture is cooled to 37°C before adding the starter culture. A fermentation process of 20 hours will then take place before the sour dough is cooled to about 4°C. The pH value of the sour dough at the time of injection is 3.4 and the acid equivavlent 14.7 (ml 0.1 N NaOH/10 g sour dough) and the number of viable bacteria 2xlO ⁸ CFU/g. Bread is baked of the type "rye bread" and sliced. Then about 18 ml sour dough is injected into the bread. After 20 hours the viable count is about 7x10 ⁷ CFU/g bread and after storage for nine days at ambient temperature (about 22-25 ⁰ C) about 3x10 ⁸ CFU/g bread.

Example 4, Preparation of probiotic bread with Lactobacillus rhamnosus 271 Sour dough is prepared using the probiotic starter culture L. rhamnosus 271

(DSM 6594), wherein a mixture of water, rye flour and an enzyme mixture (San Super 240 L (Novo Nordisk A/S, Bagsvaerd, Denmark)) is blended and heated in two steps to about 90°C. After a holding time of 60 minutes the mixture is cooled to 37°C before adding the starter culture. A fermentation process of 20 hours will then take place before the sour dough is cooled to about 4°C. The pH value of the sour dough at the time of injection is 3.4 and the acid equivavlent 12.5 (ml 0.1 N NaOH/10 g sour dough) and the number of viable bacteria 2x10 ⁹ CFU/g. Bread is baked of the type "whole-meal bread". Then about 30 ml sour dough is injected into the bread. After 24 hours the viable count is about 3x10 ⁸ CFU/g bread and

after storage for seven days at ambient temperature (about 22-25°C) about 6x10 ⁸ CFU/g bread.

Example 5. Preparation of probiotic bread with Lactobacillus plantarum SP 185 Sour dough is prepared using the probiotic starter culture L plantarum SP 158

/DSM 18789), wherein a mixture of water, sifted rye flour and an enzyme mixture (San Super 240 L (Novo Nordisk A/S, Bagsvaerd, Denmark)) is blended and heated in two steps to about 90°C. After a holding time of 60 minutes the mixture is cooled to 37°C before adding the starter culture. A fermentation process of 20 hours will then take place before the sour dough is cooled to about 4°C. The pH value of the sour dough at the time of injection is 3.4 and the acid equivalent 13.9 (ml 0.1 N NaOH/10 g sour dough) and the number of viable bacteria 1x10 ⁹ CFU/g. Bread is baked of the type "sifted rye bread". Then about 20 ml sour dough is injected into the bread. After 24 hours the viable count is about 1x10 ⁸ CFU/g bread and after storage for seven days at ambient temperature (about 22-25 ⁰ C) about 3xlO ⁸ CFU/g bread.

CLINICAL TRIAL

A clinical trial was performed aiming at studying the colonisation of the bacterium L. plantarum 299v in human intestines by studying the presence thereof in faeces after ingestion of the bacterium via a sour dough bread prepared in accordance with Example 3. 32 healthy men an women at the age of between 18 and 65 years took part in the study. The test product was a bread containing wheat, water, rye, syrup, yeast, sour dough from rye and wheat, fat, amylases and viable L. plantarum 299v. The daily intake was 120 g (3 slices) containing in total 1-2x10 ¹⁰ CFU/day. The sliced bread was ingested in the morning and evening. During the test period the test persons were not allowed to eat other products containing probiotic bacteria. The average intake of L. plantarum 299v was 2.3x10 per day. The total studying period was 5 weeks, during which the first period of 2 weeks did not comprise any consumption of the bread, the following 2 weeks involved that the test persons ingested three slices of bread per day (three slices). The final week involved no consumption of bread. The study was accepted by the ethical committee at the faculty of medicine at the university of Lund.

The faeces samples were analysed as to the content of lactobacilli (Rogosa agar, anaerobic, 3 days at 37°C) and L. plantarum 299v was identified by RAPD (Randomly Amplified Polymorphic DNA) according to Johansson et al., Lett. Appl. Microbiol. 1995;21:155-159. The results of the assay are stated in Table 3 below.

Table 3. log ₁₀ cfu/g in faeces at start and after ingestion of bread for two weeks (n=32) (median (min-max)). Comparison with before administration within the group

^a 24 of32 < 3.0 ^b l of32 < 3.0

Wilcoxon sign rank test (*p<0.05, **p<0.01, ***p<0.001).

A significant increase of the total number of lactobacilli and L. plantarum 299v was observed after ingestion of the bread for two weeks and the result is comparable to the result from other studies wherein the same number of L. plantarum 299v has been administered (Johansson et al., Int. J. Food Microiol. 42:29-38; Nobaek et al., Am. J. Gastroenterol. 95:1231-1238).